Cloning sequencing and characterization of two cell death genes and uses therefor

ABSTRACT

Described herein are genes shown to be essential for programmed cell death in  C. elegans , their encoded products (RNA and polypeptides), antibodies directed against the encoded polypeptides; probes for identifying structurally related genes and bioassays for identifying functionally related cell death genes from various organisms; methods and agents for altering (increasing or decreasing) the activity of the cell death-genes and, thus, of altering cell death; and uses therefor. Specifically, two genes shown to be essential for almost all of the cell deaths which occur in the development of  C. elegans , referred to as ced-3 and ced-4, have been cloned, sequenced and characterized.

RELATED APPLICATION

This application is a divisional of U.S. Ser. No. 08/984,178, FILED Dec.3, 1997, now abandoned, which is a continuation of U.S. Ser. No.08/287,669, filed Aug. 9, 1994, now abandoned, which is a divisional ofU.S. Ser. No. 07/979,638, filed Nov. 20, 1992, now abandoned, which is acontinuation-in-part of U.S. Ser. No. 07/897,788 now abandoned, entitled“Cloning, Sequencing and Characterization of Two Cell Death Genes andUses Therefor” by H. Robert Horvitz, Junying Yuan, and Shai Shaham,filed Jun. 12, 1992. The teachings of U.S. Ser. No. 07/897,788 areincorporated by reference.

GOVERNMENT FUNDING

Work described herein was supported by grants GM24663 and GM24943 fromthe U.S. Public Health Service. The U.S. Government has certain rightsin the invention.

BACKGROUND

Cell death is a fundamental aspect of animal development. Many cells dieduring the normal development of both vertebrates (Glucksmann, Biol.Rev. Cambridge Philos. Soc. 26:59-86 (1951)) and invertebrates (Truman,Ann. Rev. Neurosci. 7:171-188 (1984)). These deaths appear to functionin morphogenesis, metamorphosis and tissue homeostasis, as well as inthe generation of neuronal specificity and sexual dimorphism (reviewedby Ellis et al., Ann. Rev. Cell Biol. 7:663-698 (1991)). Anunderstanding of the mechanisms that cause cells to die and that specifywhich cells are to live and which cells are to die is essential for anunderstanding of animal development.

The nematode Caenorhabditis elegans is an appropriate organism foranalyzing naturally-occurring or programmed cell death (Horvitz et al.,Neurosci. Comment. 1:56-65 (1982)). The generation of the 959 somaticcells of the adult C. elegans hermaphrodite is accompanied by thegeneration and subsequent deaths of an additional 131 cells (Sulston andHorvitz, Dev. Biol. 82:110-156 (1977); Sulston et al., Dev. Biol.100:64-119 (1982)). The morphology of cells undergoing programmed celldeath in C. elegans has been described at both the light and electronmicroscopic levels (Sulston and Horvitz, Dev. Biol. 82:100-156 (1977);Robertson and Thomson, J. Embryol. Exp. Morph. 67:89-10 100 (1982)).

Many genes that affect C. elegans programmed cell death have beenidentified (reviewed by Ellis et al., Ann. Rev. Cell Biol. 7:663-698(1991)). The activities of two of these genes, ced-3 and ced-4, arerequired for the onset of almost all C. elegans programmed cell deaths(Ellis and Horvitz, Cell 44:817-829 (1986)). When the activity of eitherced-3 or ced-4 is eliminated, cells that would normally die insteadsurvive and can differentiate into recognizable cell types and evenfunction (Ellis and Horvitz, Cell 44:817-829 (1986); Avery and Horvitz,Cell 51:1071-1078 (1987); White et al., Phil. Trans. R. Soc. Lond. B.331:263-271 (1991)). Genetic mosaic analyses have indicated that theced-3 and ced-4 genes most likely act in a cell autonomous manner withindying cells, suggesting that the products of these genes are expressedwithin dying cells and either are cytotoxic molecules or control theactivities of cytotoxic molecules (Yuan and Horvitz, Dev. Biol.138:33-41 (1990)).

SUMMARY OF THE INVENTION

This invention relates to genes shown to be essential for programmedcell death, referred to herein as cell death genes, to their encodedproducts (RNA and polypeptides), and to antibodies directed against theencoded polypeptides. Methods and probes for identifying and screeningfor other cell death genes, including those of vertebrates as well asinvertebrates, and possibly, microbes and plants, are described. Agentswhich mimic or affect the activity of cell death genes and methods foridentifying these agents are also described. Bioassays which detect theactivity of cell death genes and which are useful for identifying celldeath genes, for testing the effect of mutations in cell death genes,and for identifying agents which mimic or affect the activity of celldeath genes are also provided. This invention further relates to methodsfor altering (increasing or decreasing) the activity of the cell deathgenes or their encoded products in cells and, thus, for altering theproliferative capacity or longevity of a cell population or organism.

Specifically, the ced-3 and ced-4 genes of the nematode C. elegans havebeen identified, sequenced, and characterized. These genes have beenshown to be required for almost all the programmed cell deaths whichoccur during development in C. elegans. Thus, two cell death genes andtheir encoded products (RNA, polypeptide) are now available for avariety of uses.

As described herein, the ced-3 and ced-4 genes can be used to identifystructurally related genes from a variety of sources. Some of theserelated genes are likely to also function as cell death genes.Structural comparison of related cell death genes, as well as mutationalanalysis, can provide insights into functionally important regions orfeatures of call death genes and gene products. This information isuseful in the design of agents which mimic or which alter the activityof cell death genes.

This invention further provides methods and agents for altering(increasing or decreasing) the occurrence of cell death in a cellpopulation or organism. Methods and agents, described herein, whichdecrease cell death are potentially useful for treatment (therapeuticand preventive) of disorders and conditions characterized by celldeaths, including myocardial infarction, stroke, traumatic brain injury,degenerative diseases (e.g., Huntington's disease, amyotrophic lateralsclerosis, Alzheimer's disease, Parkinson's disease, and Duchenne'smuscular dystrophy), viral and other types of pathogenic infection(e.g., human immunodeficiency virus, HIV), aging and hair loss. Methodsand agents which increase cell death are also provided and arepotentially useful for reducing the proliferation or size of cellpopulations, such as cancerous cells, cells infected with viruses (e.g.,HIV) or other infectious agents, cells which produce autoreactiveantibodies and hair follicle cells. Such methods and agents may also beused to incapacitate or kill undesired organisms, such as pests,parasites, and recombinant organisms.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the genomic organization and nucleotide sequence (Seq. ID#1) of ced-4 and deduced amino acid sequence (Seq. ID #2). The genomicsequence of the ced-4 region was obtained from plasmid C10D8-5, whichrescues the ced-4 mutant phenotype. Two likely transcriptional startsites are marked with downward arrows. The start of the cDNA is markedwith a solid arrowhead. The positions of eight ced-4 mutations areindicated by upward arrows. Numbers on the sides indicate nucleotidepositions, beginning at the start of C10D8-5. Numbers under the aminoacid sequence indicate codon positions. Vertical lines betweennucleotides indicate splice junctions.

FIG. 2 shows the genomic structure of the ced-4 gene and positions ofced-4 mutations. The sizes of exons and introns are indicated in basepairs (bp). The downward arrows indicate the positions of the Tc4insertion in the ced-4 (n1416) mutant and of eight EMS-induced mutationsof ced-4. The arrow pointing right indicates the direction oftranscription. The solid arrowhead indicates the translation initiationsite. The open arrowhead indicates the ochre termination codon.

FIG. 3 shows the sequence similarities between the Ced-4 protein andsome calcium-binding proteins. The consensus sequence of thecalcium-binding loop is shown at the top. The positions indicated by X,Y, Z, −X, and −Z correspond to vertices of an octahedron. The numbersabove the X, Y, Z, −X and −Z correspond to the positions of the residueswithin the 29 amino acid EF-hand sequence. Amino acids are indicated bythe single letter code. O, amino acid with an oxygen containing sidechain. *, non-conserved amino acid. Position Y, Z, and −X can be anyamino acid with oxygen-containing side chains. Position X is usuallyaspartic acid, and position −z is usually glutamic acid. Conserved aminoacids are shown in bold-face. Deviations from the EF-hand consensus areunderlined. The EF-hand sequences listed correspond to the following SEQID NO.s: ced-4 sequence 1 (SEQ ID NO.:30); ced-4 sequence 2 (SEQ IDNO.:31); Parvalbumin (carp) (SEQ ID NO.:3), (hake) (SEQ ID NO.:4), (ray)(SEQ ID NO.:5); SCBP (SEQ ID NO.:6), ICaBP (bovine first and secondsequence) (SEQ ID NO.s:7 and 8, respectively; Troponin C (first throughfourth sequences) (SEQ ID NO.s:9-12, respectively; Calmodulin (SEQ IDNO.:13); Trypsinogen (SEQ ID NO.:14), Fibrinogen (SEQ ID NO.:15); Villin(SEQ ID NO.:16); and GBP (SEQ ID NO.:17).

FIG. 4 shows the nucleotide sequence (Seq. ID #18) of ced-3 and deducedamino acid sequence (Seq. ID #19). The genomic sequence of the ced-3region was obtained from plasmid pJ107, which rescues the ced-3 mutantphenotype. The likely translation initiation site is indicated by asolid arrowhead. The SL1 splice acceptor of the RNA is boxed. Thepositions of 12 ced-3 mutations are indicated. Repetitive elements inthe introns are indicated as arrows above the relevant sequence. Numberson the sides indicate nucleotide positions, beginning with the start ofpJ107. Numbers under the amino acid sequence indicate codon positions.

FIG. 5A shows the genomic structure of the ced-3 gene and the locationof the mutations. The sizes of the introns and exons are given in bp.The downward arrows indicate the positions of 12 EMS-induced mutationsof ced-3. The arrow pointing right indicates the direction oftranscription. The solid arrowhead indicates the translation initiationsite. The open arrowhead indicates the termination codon.

FIG. 5B shows the locations of the mutations relative to the exons(numbered 1-8) and the encoded serine-rich region.

FIG. 6 is a Kyte-Doolittle hydrophobicity plot of the Ced-3 protein.

FIG. 7 shows a comparison of the Ced-3 proteins of C. elegans (line 1)(SEQ ID NO.:19) and related nematodes, C. briggsae (line 2) (SEQ IDNO.:20) and C. vulgaris (line 3) (SEQ ID NO.21). The conserved aminoacids are indicated by “.”. Gaps inserted in the sequence for thepurpose of alignment are indicated by “_”.

FIG. 8 shows a restriction site map of the ced-4 region and the relativepositions of plasmid C10D8-5, plasmid insert pn1416, and threetranscripts encoded by the region.

FIG. 9 shows physical and genetic maps of the ced-3 region on chromosomeIV.

FIG. 10 summarizes experiments to localize ced-3 within C48D1.Restriction sites of plasmid C48D1 and subclone plasmids are shown.ced-3 activity was scored as the number of cell corpses in the head ofL1 young animals. ++, the number of cell corpses above 10. +, the numberof cell corpses below 10 but above 2. −, the number of cell corpsesbelow 2.

DETAILED DESCRIPTION OF THE INVENTION

The ced-3 and ced-4 genes of C. elegans have been shown to be requiredfor almost all programmed cell deaths in C. elegans development (Ellisand Horvitz, Cell 44:817-829 (1986)). The present work describes thecloning, sequencing and characterization of these genes. As a result ofthis work, two genes whose activities are required for cell death,referred to herein as cell death genes, and their encoded products (RNA,polypeptide) are available for a variety of uses. Described below arethe cloning and characterization of the C. elegans ced-4 and ced-3genes, methods and probes for identifying structurally related genes,methods for identifying cell death genes from a variety of organisms,methods for identifying agents which mimic or which affect the activityof cell death genes, and methods and agents for altering cell deathactivity and thus, for altering the occurrence of cell death in a cellpopulation or organism.

The activity of a cell death gene is intended to include the activity ofthe gene itself and of the encoded products of the gene. Thus, agentsand mutations which affect the activity of a gene include those whichaffect the expression as well as the function of the encoded RNA andprotein. The agents may interact with the gene or with the RNA orprotein encoded by the gene, or may exert their effect more indirectly.

The ced-4 Gene

The cloning, sequencing and characterization of the C. elegans ced-4gene are described in Example 1. Genomic clones were obtained from aced-4 mutant allele. generated by transposon tagging. A subclonecontaining as little as 4.4 kb of wild-type genomic DNA was shown tocomplement the ced-4 mutant phenotype (see Table 1; tables are locatedat the end of the Detailed Description).

A 2.2 kb mRNA was identified as the ced-4 transcript. The transcript wasshown to be present at normal levels in a ced-3 mutant, suggesting thatced-3 is not a transcriptional regulator of ced-4 gene expression.Furthermore, the 2.2 kb transcript was shown to be expressed primarilyduring embryogenesis. This is consistent with the observation that 113of the 131 programmed cell deaths in C. elegans are embryonic (Sulstonand Horvitz, Dev. Biol. 82:110-156 (1977);

Sulston et al., Dev. Biol. 100:64-119 (1983)).

cDNA clones were further obtained and sequenced. Analysis of the cDNAand its encoded product indicates that the putative Ced-4 protein is 549amino acids in length (FIG. 1; Seq. ID #2) and about 62,877 in relativemolecular mass. The Ced-4 protein is highly hydrophilic, with apredicted pI of 5.12; there are no obvious transmembrane regions. Thelongest hydrophobic region is a segment of 12 amino acids from residues382 to 393.

Sequence analysis of the ced-4 genomic clone and comparison with thecDNA sequence revealed that the ced-4 gene contains 7 introns with sizesranging from 44 bp to 557 bp (FIG. 2).

The nucleotide sequences of eight EMS-induced ced-4 mutations were alsodetermined. Of the eight mutations, one results in a single amino acidsubstitution and the other seven appear to prevent either ced-4 RNAsplicing or completion of Ced-4 protein synthesis (FIG. 2 and Table 2).These seven mutations establish the null phenotype of the ced-4 gene,confirming that ced-4 function is not essential for viability.

Two regions of the inferred Ced-4 protein have sequence similarity toknown calcium-binding domains (Kretsinger, Cold Spring Harbor Symp.Quant. Biol. 52:499-510 (1987)), suggesting that Ced-4 activity andhence, programmed cell death may be modulated by calcium (see FIG. 3 andExample 1). Calcium has been implicated as an essential mediator of celldeath in other organisms under a variety of conditions. For example,extracellular calcium is required for glucocorticoid-induced thymocytedeath (Cohen and Duke, J. Immunol. 132:38-42 (1984)), for the deaths ofadult rat hepatocytes induced by certain toxins in vitro (Schanne etal., Science 206:700-702 (1979)), for agonist-induced muscledegeneration in mice (Leonard and Salpeter, J. Cell Biol. 82:811-819(1979)) and for neuronal cell death caused by oxygen deprivation orexcitotoxicity (Coyle et al., Neurosci. Res. Prog. Bull. 19:331-427(1981); Choi, J. Neurosci. 7:369-379 (1987), Choi, Trends Neurosci.11:465-469 (1988)). It is possible that programmed cell death isinitiated during C. elegans development by an increase in intracellularcalcium, which activates the Ced-4 protein to become cytotoxic. On theother hand, certain cells seem to be protected against cell death bycalcium (e.g., Koike et al., Proc. Natl. Acad. Sci. USA 86:6421-6425(1989); Collins et al., J. Neurosci. 11:2582-2587 (1991)), suggestingthat increases in intracellular calcium levels may inhibit the activityof the Ced-4 protein and thereby prevent programmed cell death.

The level of the ced-4 transcript in eggs is about 20% that of the actin1 transcript, which is relatively. abundant (Edwards and Wood, Dev.Biol. 97:375-390 (1983)). This level seems higher than might be expectedif ced-4 were expressed only in dying cells, since in an embryo thereare usually no more than two or three cells dying at the same time.These considerations suggest that ced-4 might be transcribed not only indying cells but in other cells as well. Perhaps Ced-4 activity, at leastduring embryonic development, is regulated at a post-transcriptionallevel. For example, the Ced-4 protein might have to interact with otherproteins or other factors (such as calcium) to cause cell death. Sincethe ced-3 gene is also essential for programmed cell death in C.elegans, one possibility is that the activity of the Ced-4 protein isdependent upon ced-3 function.

The ced-3 Gene

The cloning, sequencing and characterization of the ced-3 gene aredescribed in Example 2. The ced-3 gene was cloned by mapping DNArestriction fragment length polymorphisms (RFLPs) and chromosomewalking. A 7.5 kb fragment of genomic DNA was shown to complement ced-3mutant phenotypes. A 2.8 kb transcript was further identified. The ced-3transcript was found to be most abundant in embryos, but was alsodetected in larvae and young adults, suggesting that ced-3 is not onlyexpressed in cells undergoing programmed cell death.

A 2.5 kb cDNA corresponding to the ced-3 mRNA was sequenced. The genomicsequence was also determined (FIG. 4; Seq. ID #18) and a comparison withthe cDNA sequence revealed that the ced-3 gene has 8 introns which rangein size from 54 to 1195 bp (FIG. 5A). The four largest introns as wellas sequences 5′ of the start codon contain repetitive elements, some ofwhich have been previously characterized in non-coding regions of otherC. elegans genes such as fem-1 (Spence et al., Cell 60:981-990 (1990)),lin-12 (J. Yochem, personnal communication), and myoD (Krause et al.,Cell 63:907-919 (1990)). The transcriptional start site was also mapped,and the ced-3 transcript was found to be trans-spliced to a C. eleganssplice leader, SL1.

Twelve EMS-induced ced-3 alleles were also sequenced. Eight of themutations are missense mutations, two are nonsense mutations, and twoare putative splicing mutations (Table 3). The molecular nature of thesemutations, together with results of genetic and developmental analysesof nematodes homozygous for these mutations, indicate that, like ced-4,ced-3 function is not essential to viability. In addition, 10 out of the12 mutations are clustered in the C-terminal region of the gene (FIG.5B), suggesting that this portion of the encoded protein may beimportant for activity.

The ced-3 gene encodes a putative protein of 503 amino acids (FIG. 4;Seq. ID #19). The protein is very hydrophilic and no significantlyhydrophobic region can be found that might be a transmembrane domain(FIG. 6). One region of the ced-3 protein is very rich in serine.Sequence comparison of two additional ced-3 genes from relatednematodes, C. briggsae and C. vulgaris, suggests that the exact sequencein this serine-rich region may not be important but that the serine-richfeature is (FIG. 7; Seg. ID #19-21). This hypothesis is supported by theanalysis of ced-3 mutations: none of 12 EMS-induced ced-3 mutations isin the serine-rich region (FIG. 5B).

The conservation of the serine-rich feature among the ced-3 genes ofdifferent nematodes suggests that the serine-rich region may act insemi-specific protein-protein interactions, similar to acid blobs intranscription factors and basic residues in nuclear localizationsignals. In all these cases, the exact primary sequence is notimportant.

It is possible that the serine residues in the Ced-3 and Ced-4 proteinsmay be targets for a Ser/Thr kinase, and that the activity of theseproteins may be regulated post-translationally by proteinphosphorylation. McConkey et al. (J. Immunol., 145:1227-1230 (1990))have shown that phorbol esters, which stimulate protein kinase C, canblock the death of cultured thymocytes induced by exposure to Ca⁺⁺ionophores or glucocorticoids (Wyllie, Nature 284:555-556 (1980); Wyllieet al., J. Path. 142:67-77 (1984)). It is possible that protein kinase Cmay inactivate certain cell death proteins by phosphorylation, and thus,inhibit cell death and promote cell proliferation. Several agents thatcan elevate cytosolic cAMP levels have been shown to induce thymocytedeath, suggesting that protein kinase A may also play a role inmediating thymocyte death. Further evidence suggests that abnormalphosphorylation may play a role in the pathogenesis of certaincell-degenerative diseases. For example, abnormal phosphorylation of themicrotubule-associated protein Tau is found in the brains of Alzheimer'sdisease and Down's syndrome patients (Grundke-Iqbal et al., Proc. Natl.Acad. Sci. USA 83:4913-4917 (1986); Flament et al., Brain Res. 516:15-19(1990)). Thus, it is possible that phosphorylation may have a role inregulating programmed cell death in C. elegans. This is consistent withthe fairly high levels of ced-3 and ced-4 transcripts which suggest thattranscription regulation alone may be insufficient to regulateprogrammed cell death.

Structurally and Functionally Related Genes

As a result of the work described herein, it is possible to identifygenes which are structurally and/or functionally related to ced-3 orced-4. Such genes are expected to be found in a variety of organisms,including vertebrates (e.g., mammals and particularly humans),invertebrates (e.g., insects), microbes (e.g., yeast) and possiblyplants. Structurally related genes refer herein to genes which have somestructural similarity to the nucleotide sequences (genomic or cDNA) ofone or both of the ced-3 or ced-4 genes, or whose encoded proteins havesome similarity to one or both of the amino acid sequences of the Ced-3or Ced-4 proteins. Functionally related genes refer to genes which havesimilar activity to that of ced-3 and ced-4 in that they cause celldeath. Such genes can be identified by their ability to complement ced-3or ced-4 mutations in bioassays, as described below.

Previous studies are consistent with the hypothesis that genes similarto the C. elegans ced-3 and ced-4 genes may be involved in the celldeaths that occur in both vertebrates and invertebrates. Some vertebratecell deaths share certain characteristics with the programmed celldeaths in C. elegans that are controlled by ced-3 and ced-4. Forexample, up to 14% of the neurons in the chick dorsal root ganglia dieimmediately after their births, before any signs of differentiation(Carr and Simpson, Dev. Brain Res. 2:57-162 (1982)). Genes like ced-3and ced-4 could well function in this class of vertebrate cell death. Inaddition, genes related to ced-3 and ced-4 could function in many othertypes of vertebrate cell death processes, including those involvingcells that die long after their births and those that die as a result ofstress (e.g., oxygen deprivation) or disease.

Genetic mosaic analysis has suggested that the ced-3 and ced-4 genes actwithin cells that undergo programmed cell death, rather than throughcell-cell interactions or diffusible factors (Yuan and Horvitz, Dev.Biol. 138:33-41 (1990)). Many cell deaths in vertebrates seem differentin that they appear to be controlled. by interactions with targettissues. For example, it is thought that a deprivation of target-derivedgrowth factors is responsible for vertebrate neuronal cell deaths(Hamburger and Oppenheim, Neurosci. Comment. 1:39-55 (1982)); Thoenen etal., in: Selective Neuronal Death, Wiley, N.Y., 1987, Vol. 126, pp.82-85). However, even this class of cell death could involve genes likeced-3 and ced-4, since pathways of cell death involving similar genesand mechanisms might be triggered in a variety of ways. Supporting thisidea are several in vitro and in vivo studies which show that the deathsof vertebrate as well as invertebrate cells can be prevented byinhibitors of RNA and protein synthesis, suggesting that activation ofgenes is required for these cell deaths (Martin et al., J. Cell Biol.106:829-844 (1988); Cohen and Duke, J. Immunol. 132:38-42 (1984);Oppenheim and Prevette, Neurosci. Abstr. 14:368 (1988); Stanisic et al.,Invest. Urol. 16:19-22 (1978); Oppenheim et al., Dev. Biol. 138:104-113(1990); Fahrbach and Truman, in: Selective Neuronal Death, CibaFoundation Symposium, 1987, No. 126, pp. 65-81). It is possible that thegenes induced in these dying vertebrate and invertebrate cells are celldeath genes similar to the C. elegans genes ced-3 and ced-4.

Also supporting the hypothesis that cell death in C. elegans ismechanistically similar to cell death in vertebrates is the observationthat the protein product of the C. elegans gene ced-9 is similar insequence to the human protein Bcl-2. ced-9 has been shown to preventcalls from undergoing programmed cell death during nematode developmentby antagonizing the activities of ced-3 and ced-4 (Hengartner, et al.,Nature 356:494-499 (1992)). The bcl-2 gene has also been implicated inprotecting cells against cell death. It seems likely that the genes andproteins with which ced-9 and bcl-2 interact are similar as well.

Genes which are structurally related to ced-3 or ced-4 are likely toalso act as cell death genes. Structurally, related genes can beidentified by any number of detection methods which utilize a definednucleotide or amino acid sequence or antibodies as probes. For example,nucleic acid (DNA or RNA) containing all or part of the ced-3 or ced-4gene can be used as hybridization probes or as polymerase chain reaction(PCR) primers. Degenerate oligonucleotides derived from the amino acidsequence of the Ced-3 or Ced-4 proteins can also be used. Nucleic acidprobes can also be based on the consensus sequences of conserved regionsof genes or their protein products. In addition, antibodies, bothpolyclonal and monoclonal, can be raised against the Ced-3 and/or Ced-4proteins and used as immunoprobes to screen expression libraries ofgenes.

One strategy for detecting structurally related genes in other organismsis to initially probe animals which are taxonomically closely related tothe source of the probes, for example, probing other worms with a ced-3or ced-4 probe. Closely related species are more likely to possessrelated genes or gene products which are detected with the probe thanmore distantly related organisms. Sequences conserved between ced-3 orced-4 and these new genes can then be used to identify similar genesfrom less closely related species. Furthermore, these new genes provideadditional sequences with which to probe the molecules of other animals,some of which may share conserved regions with the new genes or geneproducts but not with ced-3, ced-4, or their gene products. Thisstrategy of using structurally related genes in taxonomically closerorganisms as stepping stones to genes in more distantly relatedorganisms can be referred to as walking along the taxonomic tree.

Groups of structurally related genes, such as those obtained by usingthe above-described strategy, can be referred to as gene families.Comparison of members within a gene family, or their encodedproducts,.may indicate functionally important features of the genes ortheir gene products. Those features which are conserved are likely to besignificant for activity. Such conserved sequences can then be used bothto identify new members of the gene family and in drug design andscreening. For example, as described in Example 2, genes similar toced-3 from two other species of nematodes (C. briggsae and C. vulgaris)were identified and characterized. Serine-rich regions were found in thepolypeptides encoded by all three genes. Although the sequence of theserine-rich region was not well conserved, the number of serines wasconserved, suggesting that the serine-rich feature, but not the exactsequence of the serine-rich region, is significant for function.

Functionally important regions can also be identified by mutagenesis.For example, inactivating mutations of ced-3 were found to clusterwithin a region near the COOH-terminus (FIG. 5B), suggesting that thisregion is a functionally important domain of the Ced-3 protein. Furthermutational analyses can be carried out on the ced-3 and ced-4 genes;mutants with novel properties, as well as other regions important foractivity, may be discovered. Mutations and other alterations can beaccomplished using known methods, such as in vivo and in vitromutagenesis (see, e.g., Ausubel et al. (eds.), Current Protocols inMolecular Biology, Greene Publishing Associates and Wiley-Interscience,New York).

Bioassays and Agents Which Affect the Activity of Cell Death Genes

This invention further provides bioassays which detect the activity ofcell death genes. The bioassays can be used to identify novel cell deathgenes, to identify mutations which affect the activity of cell deathgenes, to identify genes which are functionally related to known celldeath genes, such as ced-3 or ced-4, to identify genes which interactwith cell death genes, and to identify agents which mimic or affect theactivity of cell death genes (e.g., agonists and antagonists). Forexample, the bioassays can be used to screen expression gene librariesfor cell death genes from other organisms.

In this bioassay, genes or agents are introduced into nematodes to testtheir effect on cell deaths in vivo. Wild-type, mutant, and transgenicnematodes can be used as appropriate for the effect being tested. In oneembodiment of this bioassay, transgenic nematodes are produced using acandidate cell death gene, a mutant cell death gene, or genes from anexpression library, to observe the effect of the transgene on thepattern of programmed cell deaths during development of the nematode.For example, a gene which is structurally related to ced-3 can be usedto produce a transgenic animal from a mutant nematode whichunderexpresses or expresses an inactivated ced-3 gene to see if therelated gene can complement the ced-3 mutation and is thus, functionallyas well as structurally related to ced-3. cDNA or genomic libraries canbe screened for genes having cell death activity. Genes which interactwith cell death genes to enhance or suppress their activity can also beidentified by this method.

In another embodiment of the bioassay, wild-type, mutant, or transgenicnematodes are exposed to or administered peptides and other molecules inorder to identify agents that mimic, increase, or decrease the activityof a cell death gene. For example, wild-type animals can be used to testagents that inactivate or antagonize the activity of ced-3 or ced-4 andhence, decrease cell deaths, or that activate or enhance ced-3 or ced-4activity and increase cell deaths. Mutant animals in which ced-3 orced-4 is inactivated can be used to identify agents or genes which mimicced-3 or ced-4 in causing cell deaths. Mutant animals in which ced-3 orced-4 is overexpressed or constitutively activated can similarly be usedto identify agents that prevent ced-3 or ced-4 from causing cell death.Transgenic animals in which a wild-type or mutant form of an exogenouscell death gene causes excess cell deaths due to overexpression orhyperactivity can be used to identify agents that inactivate or inhibitthe activity of the transgene. Similarly, transgenic animals in which awild-type or mutant form of an exogenous cell death gene isunderexpressed or inactive can be used to identify agents that activateor increase its activity. Test molecules can be introduced intonematodes by microinjection, diffusion, ingestion, shooting with aparticle gun, or other method.

Mutated cell death genes with novel properties may be identified by theabove bioassay. For example, constitutively activated or hyperactivecell death genes may be isolated which may be useful as agents toincrease cell deaths. Mutations may also produce genes which do notcause cell death but which antagonize the activity of the wild-typegene.

Agents can be obtained from traditional sources, such as extracts (e.g.,bacterial, fungal or plant) and compound libraries, or by newer methodsof rationale drug design. Information on functionally important regionsof the genes or gene products, gained by sequence and/or mutationalanalysis, as described above, may provide a basis for drug design. Theactivity of the agents can be verified both by in vivo bioassays usingnematodes which express various forms of ced-3, ced-4, or related genes,as described above, and by in vitro systems, in which the genes areexpressed in cultured cells, or in which isolated or synthetic geneproducts are tested directly in biochemical experiments. The agents mayinclude all or portions of the ced-3, ced-4, or related genes, mutatedgenes, and all or portions of the gene products (RNA, includingantisense RNA, and protein), as well as nucleic acid or proteinderivatives, such as oligonucelotides and peptides, peptide andnon-peptide mimetics, and agonists and antagonists which affect theactivity or expression of the cell death genes. The acents can also beportions or derivatives of genes or gene products which are not celldeath genes but which regulate the expression of, interact with, orotherwise affect the function of cell death genes or gene products.

Uses of the Invention

Using the above-described probes and bioassays, the identification andexpression of ced-3, ced-4 or related cell death genes in culturedcells, tissues, and whole organisms can be studied to gain insights intotheir role in development and pathology in various organisms. Forexample, the detection of abnormalities in the sequence, expression, oractivity of a cell death gene or gene product may provide a usefuldiagnostic for diseases involving cell deaths.

This invention further provides means of altering or controlling theactivity of a cell death gene in a cell, and, thus, affecting theoccurrence of cell death. Activity of the cell death gene can be alteredto either increase or decrease cell deaths in a population of cells and,thus, affect the proliferative capacity or longevity of a cellpopulation, organ, or entire organism.

Agents which act as inactivators or antagonists of the activity ofced-3, ced-4, or other cell death genes can be used to prevent ordecrease cell deaths. Such agents are useful for treating (i.e., forboth preventive and therapeutic purposes) disorders and conditionscharacterized by cell deaths, including neural and muscular degenerativediseases, stroke, traumatic brain injury, myocardial infarction, viral(e.g., HIV) and other types of pathogenic infections, as well as celldeath associated with normal aging and hair loss. The agent can bedelivered to the affected cells by various methods appropriate for thecells or organs being treated, including gene therapy. For example,anti-sense RNA encoded by all or a part of a cell death gene which iscomplementary to the mRNA can be delivered to a population of cells byan appropriate vector, such as a retroviral or adenoviral vector, or anantagonist of cell death activity can be infused into a wound area tolimit tissue damage.

Methods and agents which cause or increase cell deaths are also useful,for example, for treating disorders characterized by an abnormally lowrate or number of cell deaths or by excessive cell growth, such asneoplastic and other cancerous growth. Such methods and agents are alsouseful for controlling or eliminating cell populations, such as cellsinfected with viruses (e.g., HIV) or other infectious agents, cellsproducing autoreactive antibodies, and hair follicle cells. in addition,methods and agents which increase cell death can be used to kill orincapacitate undesired organisms, such as pests, parasites andgenetically engineered organisms. All or portions of ced-3, ced-4, orrelated cell death genes, active mutant genes, their encoded products,agents which mimic the activity of cell death genes, and activators andagonists of cell death genes can be used for this purpose.

For example, cell death genes can be used to kill cells infected withthe human immunodeficiency virus (HIV), and thus, prevent or limit HIVinfection in an individual. A recombinant gene can be constructed, inwhich a cell death gene is under the control of a viral promoter whichis specifically activated by a viral protein; the recombinant gene isintroduced into HIV infected cells. HIV-infected cells containing theviral activator protein would express the cell death gene product and bekilled, and uninfected cells would be unaffected.

Alternatively, an antagonist of ced-3 or ced-4 activity (such asantisense RNA) can be expressed under the control of a viral-specificpromoter and in this way, be used to prevent the cell death associatedwith viral (e.g., HIV) infection.

In another example, cell death genes can be used as suicide genes forbiological containment purposes. Genetic engineering of suicide genesinto recombinant organisms has been reported in bacteria (GeneticEngineering News, November 1991, p. 13): suicide genes were engineeredto be expressed simultaneously with the desired recombinant gene productso that the recombinant bacteria die upon completion of their task. Thepresent invention provides suicide genes which are useful in a varietyof organisms in addition to bacteria, for example in insects, fungi, andtransgenic rodents. Suicide genes can be constructed by placing thecoding sequence of an exogenous cell death gene or an agonist of anendogenous cell death gene of the organism in an expression vectorsuitable for the organism.

In addition, agents which increase cell death are useful as pesticides(e.g., anthelminthics, nematicides). For example, many nematodes arehuman, animal, or plant parasites. ced-3, ced-4, or other nematode celldeath genes, their gene products, mimetics, and agonists can be used toreduce the nematode population in an area, as well as to treatindividuals already infected with the parasite or protect individualsfrom infection. A transgenic plant or animal carrying a constitutivelyactivated ced-3 gene, ced-4 gene, or other cell death gene specific tonematodes can be protected from nematode infection in this way.

The subject invention will now be illustrated by the following examples,which are not intended to be limiting in any way.2

EXAMPLE 1 CLONING, SEQUENCING AND CHARACTERIZATION OF THE CED-4 GENEMATERIALS AND METHODS

General Methods and Strains

Techniques used for the culturing of C. elegans were essentially asdescribed by Brenner (Genetics 77:71-94 (1974)). All strains were grownat 20° C. DNA was prepared from worms grown on Petri dishes containingagarose seeded with E. coli strain HB101. RNA was prepared from masscultures grown in liquid. Usually, the bacterial pellet from a 2 Lovernight culture of E. coli HB101 grown in superbroth (12 gBacto-tryptone, 24 g yeast extract, 8 ml 50% glycerol, 900 ml H₂O; afterautoclaving, 100 ml 0.17 M KH₂HPO₄ and 0.72 K₂HPO₄ were added) wasresuspended in 500 ml S basal medium (Brenner, 1974 supra), and wormswere added from one or two 10 cm Petri dishes in which the bacteriallawns had just been consumed. Worms were harvested about 4-5 days laterby centrifugation and washed in M9 buffer (Brenner, 1974 supra). Theyield was about 5-10 ml of packed worms.

Nomarski differential interference contrast microscopy was used toexamine individual cells in living nematodes (Sulston and Horvitz, Dev.Biol. 82:110-156 (1977)). Methods for scoring the Cad phenotype ofced-1, ced-4 and ced-1; ced-4 double mutants have been described byEllis and Horvitz, (Cell 44:817-829 (1986)) and by Yuan and Horvitz,(Dev. Biol. 138:33-41 (1990)).

The wild-type parent of all mutant strains used in these experiments wasC. elegans variety Bristol strain N2 (Brenner, 1974 supra). The geneticmarkers used are listed below. These markers have been described(Brenner, 1974 supra; Hodgkin et al., in: The Nematode Caenorhabditiselegans, Wood and the Community of C. elegans Researchers (eds.), ColdSpring Harbor Laboratory, New York, 1988, pp. 491-584; Finney et al.,Cell 55:757-769 (1988)). The strain TR679 carries the mutatormut-2(r459) (Collins et al., Nature 328:726-728 (1987)). The ced-4alleles n1894, n1920, n1947, n1948, n2247, and n2273 were characterizedin the present work. Genetic nomenclature follows the standard systemfor C. elegans (Horvitz et al., Mol. Gen. Genet. 275:129-133 (1979)):

-   -   LG I: ced-1(e1735), unc-54(r323)    -   LG III: unc-86(n1351), ced-4(n1162, n1416, n1894, n1920, n1947,        n1948, n2247, n2273, n1416 n1712, n1416 n1713), unc-79(e1068),        dpy-17 (e164)    -   LG IV: unc-31(e928), ced-3(n717)    -   LG V: egl-1(n986), unc-76(e911)        Genomic Libraries

A 4-6 kb size-selected phage library was constructed from ced-4(n1416)DNA as follows. Genomic DNA was digested with HindIII and run on alow-melting agarose gel. DNA migrating within the 4-6 kb size range wasexcised, and the low-melting agarose was removed by phenol extractionand precipitation (Maniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory (1983)). These DNA fragments wereligated to HindIII-digested DNA from phage λNM1149 (Murray, Phage Lambdaand Molecular Cloning, Cold Spring Harbor Laboratory, 1983, pp.395-432). The product DNA was packaged with packaging extract fromPromega. This library had a total of 140,000 plaque-forming units (pfu),of which 70% were recombinants, as estimated from the ratio of pfu onbacteria C600hfl and C600.

The phage genomic library (provided by J. Sulston). was prepared bypartial digestion of wild-type C. elegans genomic DNA with Sau3A andcloning into the BamHI site of phage vector λ2001 (Karn et al., Gene32:217-224 (1984)).

Tc4 Probe

The Tc4 probe used for cloning the ced-4 gene and for Southern blots wasTc4-n1351, which contains a Tc4 element isolated from an unc-86(n1351)mutant strain (Finney et al., Cell 55:757-769 (1988); Yuan et al., Proc.Natl. Acad. Sci. USA 88:3334-3338 (1991)). DNA was labelled with ³²Pusing either the nick-translation procedure described by Maniatis et al.(1983 supra) or the oligo-labelling procedure described by Feinberg andVogelstein (Anal. Biochem, 132:6-13 (1983)).

RNA Preparation, Northern Blot and Primer Extension

Total C. elegans RNA was extracted using guanidine isothiocyanate (Kimand Horvitz, Genes & Dev. 4:357-371 (1990)). Poly(A)⁺ RNA was selectedfrom total RNA by a poly(dT)-column (Maniatis et al., 1983 supra). Toprepare stage-synchronized animals, eggs were obtained from gravid C.elegans adults grown at 20° C. in liquid culture. A 5-10 ml sample ofanimals was treated with 50 ml of NaOCl/NaOH solution (10 ml NaOCl, 1 gNaOH, 40 ml H₂O) for about 10 minutes with vortexing until the adultswere dissolved. Eggs were centrifuged and washed three times with M9buffer. Isolated eggs were allowed to hatch in S basal medium withoutfood for 14 hours at 20° C. with shaking. L1 larvae were collected bylow-speed centrifugation after growth on E. coli HB101 for 2 hours, L2larvae after 12 hours, L3 larvae after 24 hours, L4 larvae after 36hours and adults after 48 hours. Northern blot analysis using DNA probeswas performed essentially as described by Meyer and Casson (Genetics106:29-44 (1986)), except that RNA was transferred from the gel to theGene Screen filter (DuPont, Wilmington, Del.) by capillary action.

Quantitation of ced-4 expression during embryonic development was doneby hybridizing two duplicate northern blots with ced-4 cDNA clone SK2-2and with a genomic DNA clone for the actin 1 gene, pW-16-210, whichhybridizes to the 3′ untranslated region of the actin 1 transcript(Krause and Hirsh, in: Molecular Biology of the Cytoskeleton, Borisy etal. (eds.), Cold Spring Harbor Laboratory, 1984, pp. 287-292). The twoprobes were of the same specific activity (4×10⁸ counts/minute/μg). Theemission of β particles from the ced-4 and actin 1 bands was countedusing a β counter (Betagen, Waltham, Mass.). The readings were 7.7counts/minute for the actin 1 band and 1.4 counts/minute for the ced-4band.

The primer extension protocol was that of Sambrook et al. (MolecularCloning: A Laboratory Manual, 2nd edition, Cold Spring HarborLaboratory, 1989, pp. 7.79-7.83), using the primer ATTGGCGATCCTCTCGA(Seq. ID #22). To define the lengths of the reaction products, asequencing reaction using this primer and C10D8-5 as template was runadjacent to the product of the primer extension reaction in thesequencing gel.

Direction of Transcription

The direction of transcription was determined by hybridizing northernblots with single-stranded RNA probes. The Bluescribe plasmid containingthe insert pn1416 was linearized by digestion with either BamHI orHindIII, which cleaved at one or the other end of the insert. Thelinearized product was transcribed using T3 or T7 RNA polymerase,respectively, generating RNA from each strand. These RNA products wereused to probe Northern blots according to a protocol developed by Z. Liuand V. Ambros: Filters were prehybridized in 50% formamide, 50 mM sodiumphosphate (pH 6.5), 5×SSC, 8×Denhardt's, 0.5% SDS, 250 μg/ml salmonsperm DNA and then hybridized with probe at 55° C. and washed in 4×SSC,0.1% SDS at 60° C. 3 times for 20 minutes each and then in 2×SSC, 0.1%SDS once at 60° C. for 20 minutes. Northern blot experiments showed thatthe single-stranded RNA probe transcribed by T3 RNA polymerasehybridized to the 2.2 kb ced-4 mRNA, while the probe made by T7 RNApolymerase did not. This result indicates that the direction of thetranscription is from the BamHI site toward the HindIII site of pn1416.

Determination of DNA Sequence

For determining DNA sequences, serial deletions were made according toHenikoff (Gene 28:351-359 (1984)). DNA sequences were determined usingSequenase and protocols obtained from US Biochemicals (Cleveland, Ohio).The ced-4 DNA sequence was confirmed by sequencing both strands of cDNAand genomic DNA clones.

Cloning of the Cosmid Fragment C10D8-5

The cosmid C10D8 was digested with EcoRI. Two EcoRI fragments of 2.2 kb(r5) and 2.4 kb (r7), both of which hybridized to a mixture of ced-4cDNA subclones SK2-1 and SK2-2, were isolated. r7, which hybridized toSK2-1, which contains the 3′ half of ced-4 cDNA clone SK2, was clonedinto the EcoRI site of plasmid pBSKII (Stratagene). The EcoRI site atthe 3′ end of r7 was deleted by digesting with Styl, which cut once at0.2 kb from the 3′ end of the insert, and SalI, which cut once in thepolylinker, and then religating. The deleted r7 plasmid was linearizedwith EcoRI and ligated with EcoRI-digested r5, which hybridized toSk2-2, the 5′ half of ced-4 cDNA SK2. Clones were analyzed for thecorrect orientation of the r5 insert based on the cDNA restriction map.One such correctly oriented clone was named C10D8-5.

Microinjection and Transformation

The procedure;for microinjecting DNA into the gonad to obtain germlinetransformants was basically that of Fire (EMBO J. 5:2673-2680 (1986))with modifications introduced by J. Sulston. Cosmid DNA to be injectedwas purified twice using CsCl-gradient centrifugation (Maniatis et al.,1983 supra). Plasmid DNA to be injected was prepared by alkalineminipreps (Maniatis et al., 1983 supra). DNA was treated with RNAase A(37° C., 30 minutes) and then with proteinase K (55° C., 30 minutes),extracted with phenol and then chloroform, precipitated twice (first in0.3 M sodium acetate and then in 0.1 M potassium acetate, pH 7.2), andresuspended in 5 ul of injection buffer (Fire, 1986 supra). DNAconcentrations used for injection were 0.1-1.0 mg/ml.

All transformation experiments used a ced-1; ced-4(n1162); unc-31 strainas the recipient. The expression of the Ced-4 phenotype was quantifiedby counting the number of cell corpses in the heads of young L1 animals.The cosmid C10D8 or plasmid subclones of C10D8 were mixed with cosmidC14G10, which contains the wild-type unc-31(+) gene, at a ratio of 2:1or 3:1 to increase the likelihood that a phenotypically non-Unctransformant would contain the cosmid or plasmid being tested.Generally, 20-30 animals were injected in one experiment. Non-Unc F1progeny of injected animals were isolated three to four days later.About ½ to ⅓ of the non-Unc progeny transmitted the non-Unc phenotype totheir progeny and could be established as lines of transformants. YoungL1 non-Unc progeny of such non-Unc transformants were examined usingNomarski optics to determine the number of cell corpses present in theheads.

Ced-4 Fusion Protein and Antibody Preparation

To express a Ced-4 fusion protein in E. coli, a clone containing boththe 5′ and 3′ halves of the ced-4 cDNA (SK2-2 and SK2-1) in theexpression vector pET-5a (Rosenberg et al., Gene 56:125-135 (1987)) wasconstructed. The fusion protein expressed by this vector was expected toinclude 11 amino acids of phage T7 gene 10 protein, 5 amino acids oflinker and the 546 amino acids encoded by ced-4 cDNA SK2. The pJ76plasmid, which encodes this fusion protein, was transformed intobacterial strain BL21. ced-4 fusion protein was produced by thistransformed strain, as expected, and subjected to electrophoresis on apolyacrylamide gel. A band, with mobility equivalent to about 64×10³ Mr,specific to the transformed strain was exercised and used to immunizethree rabbits. Sera from all three rabbits tested positive on western.blots (Towbin et al., Proc. Natl. Acad. Sci. USA 76:4350-4354 (1979)).These sera were purified sing immunoblots (Harlow and Lane, Antibodies:A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).

RESULTS

Cloning of the ced-4 Gene by Transposon Taggaing

The ced-4 allele n1416 in the C. elegans strain TR679 was isolated,which carries the mutator mut-2(r459) and shows an elevated frequency oftransposition elements (Collins et al., Nature 328:726-728. (1987); Yuanet al., Proc. Natl. Acad. Sci. USA 88:3334-3338 (1991)). Theced-4(n1416) mutation is closely linked to a newly transposed copy ofthe C. elegans transposon Tc4 (Yuan et al., 1991 supra). Using Tc4 as aprobe, this novel Tc4 element and its flanking region was cloned as a 5kb HindIII fragment from a 4-6 kb size-selected ced-4(n1416) genomicphage library. A 3 kb adjacent to this Tc4 element was isolated bydigesting the 5 kb HindIII fragment with BamHI. This 3 kb fragment,called pn1416, was cloned into the Bluescribe M13+ plasmid vector(Stratagene),.

When used as a probe on Southern blots, pn1416 hybridized to a 3.4 kbHindIII fragment in DNA of wild-type (strain N2) and two non-Cedrevertants of ced-4(n1416), ced-4(n1416 n1712) and ced-4(n1416 n1713)(Yuan and Horvitz, Dev. Biol. 138:33-41 (1990)), and a 5 kb HindIIIfragment in ced-4(n1416) animals. The hybridizing band in ced-4(n1416)DNA is 1.6 kb larger than that of the wild-type or the revertants,indicating that an insertion of this size is present in the ced-4(n1416)strain and is deleted in both revertants. These observations indicatethat the Tc4 insertion in ced-4(n1416) animals is responsible for theirCed-4 mutant phenotype and suggest that pn1416 contains at least part ofthe ced-4 gene.

To isolate additional genomic DNA from the region of this Tc4 insertion,pn14l6 was used to probe a C. elegans Bristol N2 genomic DNA phagelibrary. Five phage clones with inserts of 10 to 15 kb were isolated andshown to share a 3 kb BamHI-HindIII fragment that hybridized to pn1416.These phage clones were used to identify cosmids that hybridized to themand that were members of a 600 kb contig of overlapping cosmids (Coulsonet al., Proc. Natl. Acad. Sci. USA 83:7821-7825 (1986)). By using thephage clones as probes to hybridize to Southern blots, a cosmid C10D8was identified as containing all regions of genomic DNA present in allfive phage clones and in pn1416.

The ced-4 Mutant Phenotype Can Be Rescued by a 4.4 kb DNA Fragment

To identify ced-4(+) DNA capable of complementing the Ced-4 mutantphenotype, the cosmid C10D8 was injected into the oocytes ofced-4(n1162) animals. To facilitate the identification of transgenicanimals, a mutation in the unc-31 gene, which affects locomotion, wasincluded as a marker for co-transformation (Kim and Horvitz, Genes &Dev. 4:357-371 (1990)). Cosmid C14G10, which contains the wild-typeallele of unc-31 and does not have Ced-4-rescuing activity wascoinjected with cosmid C10D8 into ced-1(e1735); unc-31(e928);ced-4(n1162) animals. The ced-1 mutation was included to facilitate thescoring of the ced-4 mutant phenotype (Ellis and Horvitz, Cell44:817-829 (1986)). Specifically, when a cell undergoes programmed celldeath in C. elegans, its corpse is quickly engulfed and destroyed by aneighboring cell (Robertson and Thomson, J. Embryol. Exp. Morph.67:89-100 (1982); Sulston et al., Dev. Biol. 100:64-119 (1983)). A ced-1mutation prevents this engulfment, allowing the cell corpse to remainintact (Hedgecock et al., Science 220:1277-1280: (1983)). Thus, in afirst or second stage (L1 or L2) ced-1 mutant larva, many cell corpsesare present and can be easily visualized using Normaski optics. ced-4mutations prevent cell death and the appearance of these corpses. Thus,suppression of the Ced-4 mutant phenotype by a wild-type ced-4 gene canbe observed and readily quantified in a ced-1 mutant background based onan increase in the number of visible cell corpses.

From one such microinjection experiment, three non-Unc animals rescuedfor the Unc-31 mutant phenotype were picked from among the F1 progeny,and from one of them a line of non-Unc transformants was obtained. Notrue-breeding non-Unc animals could be isolated from this line: about25% of the progeny of all non-Unc animals were Unc. since no inviablezygotes were observed among the progeny of these non-Unc animals, thistransformant did not carry a recessive lethal insertion mutation.Rather, it seems likely that the injected DNA was maintained as anextrachromosomal array that was segregated to only some gametes, as hasbeen reported previously for many other C. elegans transgenic strains(e.g., Stinchcomb et al., Mol. Cell Biol. 82:110-156 (1985);. Fire, EMBOJ. 5:2673-2680 (1986); Way and Chalfie, Cell 54:5-16 (1988)). Thisputative extrachromosomal array was named nEx1. Young L1 progeny ofnEx1-containing animals were examined using Nomarski optics for theCed-4 phenotype.

Young L1 ced-1 animals have an average of 23 cell corpses in the head,while ced-1 (e1735); ced-4 (n1162) animals have an average of 0.6 cellcorpses (Ellis and Horvitz, Cell 44:817-829 (1986)). Young L1 ced-1;ced-4(n1162); nEx1 animals had an average of nine cell corpses in thehead. These results indicate that cosmid C10D8 restored significant, butnot total, ced-4(+) activity in the transformants.

To delineate the ced-4 gene within C10D8, various subclones of C10D8were injected into ced-4 mutant animals and tested for their ability torescue the Ced-4 mutant phenotype (Table 1). The smallest subcloneplasmid that could rescue the ced-4 phenotype as effectively as cosmidC10D8 was a 4.4 kb fragment, called C10D8-5. C10D8-5 and theunc-31(+)-containing cosmid C14G10 were coinjected into ced-1; unc-31;ced-4(n1162) animals. Two lines of non-Unc transformants were isolated.Since these animals continued to segregate Unc animals and did notproduce inviable zygotes, both appeared to carry extrachromosomalarrays, which were designated nEx7 and nEx8. Young L1 animals from thesetransformant strains had an average of 11.5 cell corpses in their heads,indicating that plasmid C10D8-5 restored ced-4(+) activity as well asdid cosmid C10D8 (Table 1).

Identification of a ced-4 Transcript

Restriction sites of plasmid C10D8-5 (which can rescue the Ced-4phenotype) and pn1416 (which contains sequences adjacent to the Tc4insertion site) were mapped. C10D805 was found to overlap with 2 kb ofsequence in pn1416, including the Tc4 insertion site (FIG. 8).

In Northern blot experiments, both pn1416 and C10D8-5 were used to probepoly(A)⁺ RNA populations of mixed developmental stages of wild-type(strain N2), ced-4(n1416), and ced-4(n1416 n1712) and ced-4(n1416 n1713)revertant animals. pn1416 hybridized to a 2.2 kb transcript and an 0.9kb transcript in RNA from N2 animals, and a 3 kb transcript, atranscript slightly larger than the wild-type 2.2 kb transcript, and atranscript slightly smaller than the wild-type 0.9 kb transcript inced-4(n1416) animals. The 3.8 kb RNA contained Tc4 sequence (see below),suggesting that this RNA resulted from the insertion of the 1.6 kb Tc4sequence into the ced-4 sequence encoding 2.2 kb transcript. Thetranscript slightly larger than the 2.2 kb wild-type transcript did notcontain Tc4 sequence. This ced-4(n1416) RNA might have been an aberranttranscript containing sequences adjacent to the ced-4 gene: when pn1416was used as a probe, the wild-type 2.2 kb and the slightly largertranscript in this mutant were relatively similar in intensities,whereas when ced-4 cDNA clone SK2-1 was used as a probe, this mutanttranscript was not detected (see below). These observations indicatethat the ced-4(n1416) 2.2 kb transcript contains sequences from theced-4 region but does not contain sequences corresponding to at leastthe 3′ half of the ced-4 mRNA. The two revertants of ced-4(n1416),ced-4(n1416 n1712) and ced-4(n1416 n1713), contained both 2.2 kb and 0.9kb transcripts with similar sizes to the wild-type transcripts. Thus,both the 2.2 kb and the 0.9 kb transcripts were altered in ced-4(n1416)animals, and both were restored in the two non-Ced revertants.

To determine if any of the transcripts contains Tc4 sequence, theNorthern blots were probed with Tc4-n1351, which contains the 1.6 kb Tc4element present in the Tc4-induced mutant unc-86(n1351) as well as 4 kbof unc-86 sequences. Tc4-n1351 hybridized both to a 3.8 kb transcript ofthe Tc4-induced mutant ced-4(n1416) and to a 1.5 kb unc-68 transcript inboth ced-4(n1416) and N2 animals.

To determine whether one or both of the 2.2 kb and 0.9 kb transcriptsare encoded by ced-4, subclone C10D8-5, which rescued the Ced-4phenotype, was used to probe the Northern blots. C10D8-5 detected thewild-type 2.2 kb transcript, the ced-4(n1416) transcript slightly largerthan the 2.2 kb transcript, and the ced-4(n1416) 3.8 kb transcript.C10D8-5 did not hybridize to the 0.9, kb transcript, indicating thatthis transcript is unlikely to be encoded by ced-4. C10D8-5 alsodetected a 1.4 kb transcript, which was not altered by the Tc4 insertionin ced-4(n1416) animals only a 470 bp EcoRI-StuI fragment at one end ofC10D8-5 hybridized to this 1.4 kb RNA. Since C10D8-5 did not contain thecomplete coding region for this RNA, and since this RNA was unaffectedin ced-4(n1416) animals, this 1.4 kb RNA seems unlikely to be a ced-4transcript. The relationships among cosmid C10D8-5, pn1416 and the 0.9kb, 1.4 kb and 2.2 kb transcripts are summarized in FIG. 8.

On Northern blots probed with the ced-4 cDNA clone SK2-1, the level ofthe 2.2 kb transcript showed significant reduction in all threeindependently derived EMS-induced ced-4 mutants examined, stronglysupporting the hypothesis that this 2.2 kb transcript is a ced-4transcript. Total RNA from N2, ced-4(n1162), ced-4(n1416), ced-4 (n1894)and ced-4 (n1920) eggs was probed with ³²P-labelled ced-4 cDNA SK2-1. Anactin 1 probe (Krause and Hirsh, in: Molecular Biology of theCytoskeleton, Borisy et al. (eds.), Cold Spring Harbor Laboratory, 1984,pp. 287-292) was used as an internal control for the amount of RNAloaded in each lane. The ratios of the intensity of the ced-4 band tothat of actin band in N2, n1162, n1416 and n1894 were 0.5, 0.17, 0 and0.12, respectively. A Northern blot of poly(A)+ RNA fromstage-synchronized animals was probed with pn1416, which hybridizes bothto the 2.2 kb ced-4 transcript and to a 0.9 kb transcript. The 0.9 kbtranscript seems to be expressed mostly in eggs and adults. The presenceof RNA in all lanes was confirmed by loading 1/10 of each sample onanother gel and probing a Northern blot from this gel using the C.elegans actin 1 gene (Krause and Hirsh, 1984 supra). That all of thesedistinct ced-4 mutations cause reduced levels of a ced-4 transcriptcould reflect either instability of all. three mutant transcripts or arole for ced-4 in regulating its own expression.

Based upon these results, it can be concluded that the 2.2 kb RNA is aced-4 transcript. It is, not known why the 0.9 kb RNA is also altered inced-4(n1416) animals. Perhaps transcription of the 0.9 kb RNA isinitiated incorrectly as a consequence of the nearby Tc4 element.

ced-4 Expression is Primarily Embryonic

A Northern blot containing RNAs from stage-synchronized animals ofdifferent developmental stages probed with pn1416 showed that the 2.2 kbced-4 transcript was expressed primarily during embryogenesis. Thisresult is consistent with the observation that 113 of the 131 programmedcell deaths in the C. elegans hermaphrodite are embryonic (Sulston andHorvitz, Dev. Biol. 82:110-156 (1977); Sulston et al., Dev. Biol.100:64-119 (1983)). The 2.2 kb RNA was relatively abundant duringembryonic development. The 0.9 kb transcript was expressed mostly ineggs and adults. The presence of RNA in all lanes was confirmed byloading 1/10 of each sample on another gel and probing a Northern blotfrom this gel with the C. elegans actin 1 gene (Krause and Hirsh, 1984supra).

The ced-4 Transcript is Present in a ced-3 Mutant

The activities of both ced-3 and ced-4 are required for programmed celldeath (Ellis and Horvitz, Cell 44:817-819 (1986)). One possibility isthat one of these genes positively regulates the expression of theother. For this reason, a Northern blot of wild-type strain N2 andced-3(n717) poly(A)⁺ RNA was probed with pn1416. This experiment showedthat the 2.2 kb ced-4 transcript was present at an apparently normallevel in this ced-3 mutant. Thus, the activity of the ced-3 gene isunlikely to be necessary for the expression of the ced-4 2.2 kbtranscript.

Identification of ced-4 cDNA Clones

To isolate cDNA clones of ced-4, pn1416 was used to probe a C. eleganscDNA phage library made from wild-type strain N2 mixed-stage RNA (Kimand Horvitz, Genes & Dev. 4:357-371 (1990)). Two cDNA clones wereisolated. The two cDNA clones (named SK1 and SK2) hybridized to the 2.2kb ced-4 transcript. Both are about 1.8 kb in size, and both contain one0.8 kb and one 1.0 kb EcoRI fragment. These EcoRI fragments weresubcloned into plasmid vector Bluescribe M13+ (Stratagene). The twosubclones derived from SK1 were named SK-1 and SK1-2, and the twosubclones derived from SK2 were named SK2-1 and SK2-2. The restrictionmaps of the SK1- and SK2-derived clones were the same. Sequence analysisof the ends of the four cDNA subclones confirmed the equivalence of theSK1 and SK2 clones, except that SK1-2 contains a poly(A) sequence ofmore than 50 bp at its 5′ end. This poly(A) sequence is probably a cDNAcloning artifact, since SK1-2 contains the 5′ half of the cDNA (seebelow).

The ced-4 Sequence

The DNA sequence of the SK2 1.8 kb cDNA clone was. determined. Thissequence includes an open reading frame encoding 546 amino acids (FIG.1; Seq. ID #2), which is consistent with the results of Northern blotanalysis using single-stranded RNA probes. An ochre termination codon(TAA) is located in-frame near the 3′ end, indicating that the 3′ end ofthe 2.2 kb transcript is most likely included in this cDNA. The openreading frame extends to the 5′ end of the 1.8 kb cDNA, suggesting thatthis cDNA might lack the 5′ end of the ced-4 coding region.

A primer extension experiment was performed to determine the ced-4transcription initiation site(s) using the primer ATTGGCGATCCTCTCGA(Seq. ID #23) and C10D8-5 as template. A major transcriptionalinitiation site was identified at 54 bp before (5′ of) the beginning ofthe ced-4 cDNA SK2 and a minor initiation site at 54 bp after (3′ of)the beginning of this cDNA (FIG. 1). The first AUG codon after thepresumptive major start site is located at 9 bp before the beginning ofthe cDNA (FIG. 1). If this site is used to initiate protein synthesis,the Ced-4 protein would be 549 amino acids in length. The first AUGcodon after the presumptive minor start site is located at 130 bp afterthe beginning of the cDNA. If this site is used, the Ced-4 protein wouldbe 503 amino acids in length. Preliminary results using an anti-Ced-4antibody raised against a Ced-4 fusion protein showed that endogenousCed-4 protein is slightly smaller in molecular weight than a Ced-4fusion protein of 562 amino acids expressed in E. coli. Thus, most Ced-4protein is probably initiated near the start of the cDNA and ispresumably 549 amino acids in length and 62,977 in relative molecularmass. The direction of the open reading frame is consistent with thedirection of transcription, as demonstrated by probing Northern blotswith single-stranded RNA probes. The presumptive Ced-4 protein is highlyhydrophilic, with a pI of 5.12. The longest hydrophobic region is asegment of 12 amino acids from residues 382 to 393.

A Western blot of wild-type strain N2 mixed-stage, ced-4(n1416)mixed-stage, wild-type egg, and bacterially expressed protein (pJ76) wasprobed using anti-Ced-4 antibody. Ced-4 fusion protein (pJ76) was madeby cloning ced-4 cDNA SK2 into the T7 expression vector pET-5a(Rosenberg et al., Gene 56:125-135 (1987)), so that 546 amino acids ofCed-4 sequence were fused to 11 amino acids of T7 gene 10 protein and 5amino acids of linker sequence. This Ced-4 fusion protein is similar inrelative molecular mass to the endogenous Ced-4 protein, which ispresent in wild-type (N2) but missing in ced-4(n1416) animals. Theproteins phosphorylase b, 97×10³; bovine serum albumin, 66×10³ (Hirayamaet al., Biochem. Biophys, Res. Comm. 173:639-646 (1990)); and ovalbumin,43×10³, were used as molecular weight standards.

To confirm the DNA sequence obtained from the ced-4 cDNAs and to studythe structure of the ced-4 gene, the sequences of the 4.4 kb cosmidsubclone C10DS-5, the 3 kb insert pn1416, and the 2 kb HindIII-BamHIfragment that contains the Tc4 insertion in the ced-4(n1416) mutant weredetermined. Comparison of the ced-4 genomic and cDNA sequences revealedthat the ced-4 gene has seven introns of sizes ranging from 44 bp to 557bp (FIG. 2). The exon sequences of genomic clone C10D8-5 are identicalto the sequences of ced-4 cDNA SK2. Comparison of the Tc4 insertion sitein ced-4(n1416) DNA with the ced-4(+) genomic and cDNA sequencesindicated that Tc4 inserted into an exon in the ced-4 gene inced-4(n1416) animals (FIG. 2).

The DNA sequences of eight EMS-induced ced-4 alleles were alsodetermined (Table 2). One of the eight, n1948, is a missense mutation.Of the seven others, four create stop codons and three are predicted toaffect splicing of the ced-4 transcript. The positions of thesemutations are indicated in FIG. 2. These findings indicate that thephenotypes of these mutants (Ellis and Horvitz, Cell 44:817-829 (1986))result from a complete loss of ced-4 gene function. These mutationsestablish the null phenotype of the ced-4 gene, confirming that ced-4function is not essential for viability.

The Ced-4 Protein Has Two Regions Similar to Known Calcium-BindingDomains

By direct inspection, the sequence of the putative Ced-4 protein wascompared with the consensus sequence of the calcium-binding loop of theEF-hand domain (Tufty and Kretsinger, Science 187:161-171 (1975);Kretsinger, Cold spring Harbor Symp. Quant. Biol. 52:499-510 (1987);Szebenyi and Moffat, J. Biol. Chem. 26:8761-8777 (1986)). Two regions ofthe Ced-4 protein were identified that might bind calcium (FIG. 3).

The EF-hand is a 29 amino acid domain consisting of a helix-loop-helixregion, with the loop portion (residues 10-21) coordinatingcalcium-binding via the side-chain oxygens of serine, threonine,asparagine, aspartic acid, glutamine or glutamic acid. These residuesoccur at five of the vertices of an octahedron: X (position 10), Y (12),Z (14), −X (18), −Z (21). EF-hand amino acid sequences vary considerablyin the residues present in the calcium-binding loop (FIG. 3), and someEF-hand domains have only one helical region (Kretsinger, 1987 supra).The consensus sequence is shown at the top of FIG. 3. Positions Y, Z,and −X can have any of a number of amino acids which haveoxygen-containing side chains. Position X is usually aspartic acid, andposition −Z is usually glutamic acid.

The sequences of parvalbumins from carp muscle (Seq. ID #3; Nockolds etal., Proc. Natl. Acad. Sci. USA 69:581-584 (1972)), the intestinalcalcium-binding protein (ICaBP) (Seq. ID #7-8; Szebenyi et al., Nature294:327-332 (1981)), troponin C (Seq. ID #9-12; Collins et al., FEBSLett. 36:268-272 (1973)) and calmodulin (Seq. ID #13; Zimmer et al., J.Biol. Chem. 263:19,370-19,383 (1988); Babu et al., Nature 315:37-40(1985)) show canonical EF-hands. The hake and ray parvalbumins (Seq. ID#4-5; Capony et al. Eur. J. Biochem. 32:97-108 (1973)); Thatcher andPechere, Eur. J. Biochem. 75:121-132 (1977)), sarcoplasmiccalcium-binding protein (SCBP) from the protochordate Amphioxus (Seq. ID#6; Takagi et al., Biochemistry 25: 3585-3592 (1986)), trypsinogen (Seq.ID #14; Bode and Schwager, J. Mol. Biol. 98:693-717 (1975)), fibrinogen(Seq. ID #15; Doolittle, Ann. Rev. Biochem. 53:195-229 (1984); Dang etal., J. Biol. Chem. 260:9713-9719 (1985)), villin (Seq. ID #16;Hesterberg and Weber, J. Biol. Chem. 258:365-369 (1983)) andgalactose-binding protein (GBP) (Seq. ID #17; Vyas et al., Nature327:635-638 (1987)) show variations from the consensus sequence. GBPdoes not contain the helices of the EF-hand.

The potential calcium-binding loops of sequence 1 and sequence 2 arelocated at amino acids 77-88 and amino acids 292-303 of the Ced-4protein, respectively (FIG. 3). In its putative calcium-binding loop,the first potential EF-hand-like sequence of the Ced-4 protein has four(positions Y, Z, −X, −Z) of the five conserved residues withoxygen-containing side chains (shown in bold), and the fifth position(X) has a tyrosine rather than an aspartic acid; tyrosine containsoxygen in its side chain. The second potential EF-hand-like sequence ofthe Ced-4 protein has three residues (positions Z, −X, −Z) that matchthe consensus sequence, and amino acids with oxygen-containing sidechains at the other two positions. These observations suggest that thesetwo regions of the Ced-4 protein might bind calcium. Like the Ced-4protein, a number of known calcium-binding proteins, such a bovineintestinal calcium-binding protein (ICaBP) (Szebenyi and Moffat, 1986supra), rabbit troponin C (Collins et al., 1973 supra), trypsinogen andvillin (Doolittle, 1984 supra; Danget et al., 1985 supra) have onlythree or four conserved residues at these five positions (FIG. 3). TheEF-hand domains in ICaBP and troponin C have been shown by X-raycrystallography to bind calcium.

One major difference between the Ced-4 protein and the calcium-bindingloop of the EF-hand consensus sequence is at position 15. Here, the twoCed-4 sequences have a histidine and a glutamic acid, respectively;whereas most ET-hand-containing proteins have a glycine; this glycinehas been suggested to be important for the turning of the loop(Kretsinger, 1987 supra). However, a histidine is present at thisposition in a parvalbumin and an aspartic acid is present in anotherparvalbumin and also in a sarcoplasmic calcium-binding protein(Kretsinger, 1987 supra) (FIG. 3). Thus, the presence of histidine orglutamic acid at position 15 does not rule out the possibility thatthese regions bind calcium.

The calcium-binding loop (positions 10-21) of the EF-hand is thought tobe preceded (positions 1-9) and followed by alpha-helical domains(positions 22-29) (Kretsinger, 1987 supra). Since position 3 of Ced-4sequence 1 and positions 26 and 28 of Ced-4 sequence 2 are prolines,these regions might not form alpha-helices. However, the knowncalcium-binding protein galactose-binding protein (GBP) has acalcium-binding domain similar to that of the EF-hand (FIG. 3) butwithout the two helices; furthermore, position 29 of GBP is proline(Vyas et al., 1987 supra). Thus, the Ced-4 protein need not contain suchalpha-helical calcium-binding domains.

Based upon these considerations, it seems likely that the Ced-4 proteinbinds calcium or a similar divalent cation.

EXAMPLE 2 CLONING, SEQUENCING, AND CHARACTERIZATION OF THE CED-3 GENEMATERIALS AND METHODS

General Methods and Strains

The techniques used for the culturing of C. elegans were as described byBrenner (Genetics 77:71-94 (1974)). All strains were grown at 20° C. Thewild-type parent strains were C. elegans variety Bristol strain N2,Bergerac strain EM1002 (Emmons et al., Cell 32:55-65 (1983)), C.briggsae and C. vulgaris (obtained from V. Ambros). The genetic markersused are described below. These markers have been described by Brenner(1974 supra), and Hodgkin et al. (In: The Nematode Caenorhabditiselegans, Wood and the Community of C. elegans Researchers (eds.), ColdSpring Harbor Laboratory, 1988, pp 491-584). Genetic nomenclaturefollows the standard system (Horvitz et al., Mol. Gen. Genet.175:129-133 (1979)).

-   -   LG I: ced-1(e1375); unc-54(r323)    -   LG VI: unc-31(e928), unc-30(e191), ced-3(n717, n718, n1040,        n1129, n1163, n1164, n1165, n1286, n1949, n2426, n2430, n2433),        unc-26(e205), dpy-4 (e1166)    -   LG V: egl-1(n986); unc-76(e911)    -   LG X: dpy-3(e27)        Isolation of Additional Alleles of ced-3

A non-complementation screen was designed to isolate new alleles ofced-3. Because animals heterozygous for ced-3(n717) in trans to adeficiency are viable (Ellis and Horvitz, Cell 44:817-829 (1986)),animals carrying a complete loss-of-function ced-3, allele generated bymutagenesis were expected to be viable in trans to ced-3(n717), even ifthe new allele was inviable in homozygotes. Fourteen EMS mutagenizedegl-1 males were mated with ced-3 (n717) unc-26(e205); egl-1(n487);dpy-3(e27) hermaphrodites. egl-1 was used as a marker in this screen.Dominant mutations in egl-1 cause the two hermaphrodite specificneurons, the HSNs, to undergo programmed cell death (Trent et al.,Genetics 104:619-647 (1983)). The HSNs are required for normalegg-laying, and egl-1(n986) hermaphrodites, which lack HSNs, areegg-laying defective (Trent et al., 1983 supra). The mutant phenotype ofegl-1 is suppressed in a ced-3; egl-1 strain because mutations in ced-3block programmed cell deaths. egl-1 males were mutagenized with EMS andcrossed with ced-3(n717), unc-26(e205); egl-1(n487); dpy-3 (e27). Mostcross progeny were egg-laying defective because they were heterozygousfor ced-3 and homozygous for egl-1. Rare egg-laying competent animalswere picked as candidates for carrying new alleles of ced-3. Four suchanimals were isolated from about 10,000 F1 cross progeny ofEMS-mutagenized animals. These new mutations were made homozygous toconfirm that they carried recessive mutations of ced-3.

Molecular Biology

Standard techniques of molecular biology were used (Maniatis et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,1983).

Two cosmid libraries were used extensively in this work: a Sau3AIpartial digest genomic library of 7000 clones in the vector pHC79 and aSau3AI partial digest genomic library of 6000 clones in the vector pJB8(Ish-Horowicz and Burke, Nucleic Acids Res. 9:2989 (1981)).

The “right” end of MMM-C1 was cloned by cutting it with HindIII andself-ligating. The “left” end of MMM-C1 was cloned by cutting it withBglII or SalI and self-ligating.

The “right” end of Jc8 was made by digesting Jc8 with EcoRI andself-ligating. The “left” end of Jc8 was made by digesting Jc8 by SalIand self-ligating.

C. elegans RNA was extracted using guanidine isothiocyanate (Kim andHorvitz, Genes & Dev. 4:357-371 (1990)). Poly(A)⁺ RNA was selected fromtotal RNA by a poly(dT) column (Maniatis et al., 1983 supra). To preparestage-synchronized animals, worms were synchronized at differentdevelopmental stages (Meyer and Casson, Genetics 106:29-44 (1986)).

For DNA sequencing, serial deletions were made according to a proceduredeveloped by Henikoff (Gene 8:351-359 (1984)). DNA sequences weredetermined using Sequenase and protocols obtained from US Biochemicalswith minor modifications.

The Tc1 DNA probe for Southern blots was pCe2001, which contains aBergerac Tc1 element (Emmons et al., Cell 32:55-65 (1983)). Enzymes werepurchased from New England Biolabs, and radioactive nucleotides werefrom Amersham.

Primer extension procedures followed the protocol by Robert E. Kingston(In: Current Protocols in Molecular Biology, Ausubel et al. (eds.),Greene Publishing Associates and Wiley-Interscience, New York, p. 4.8.1)with minor modifications.

Polymerase chain reaction (PCR) was carried out using standard protocolssupplied by the GeneAmp Kit (Perkin Elmer). The primers used for primerextension and PCR are as follows:

-   -   Pex2: 5′ TCATCGACTTTTAGATGACTAGAGAACATC 3′ (Seq. ID #24);    -   Pex1: 5′ GTTGCACTGCTTTCACGATCTCCCGTCTCT 3′ (Seq. ID #25);    -   SL1: 5′ GTTTAATTACCCAAGTTTGAG 3′ (Seq. ID #26);    -   SL2: 5′ GGTTTTAACCAGTTACTCAAG 3′ (Seq. ID #27);    -   Log5: 5′ CCGGTGACATTGGACACTC 3′ (Seq. ID #28); and    -   Oligo10: 5′ ACTATTCAACACTTG 3′ (Seq. ID #29).        Germline Transformation

The procedure for microinjection basically follows that of A. Fire (EMBOJ. 5:2673-2680 (1986)) with modifications: Cosmid DNA was twice purifiedby CsC1-gradient. Miniprep DNA was used when deleted cosmids wereinjected. To prepare miniprep DNA, DNA from 1.5 ml overnight bacterialculture in superbroth (12 g Bacto-tryptone, 24 g yeast extract, 8 ml 50%glycerol, 900 ml H₂O, autoclaved; after autoclaving, 100 ml 0.17 MKH₂PO₄ and 0.72 M KH₂PO₄ were added) was extracted by alkaline lysismethod as described in Maniatis et al. (1983 supra). DNA was treatedwith RNase A (37°, 30 minutes) and then with protease K (55°, 30minutes), extracted with phenol and then chloroform, precipitated twice(first in 0.3 M sodium acetate and second in 0.1. M potassium acetate,pH 7.2), and resuspended in 5 μl injection buffer as described by A.Fire (1986 supra). The DNA concentration for injection is in the rangeof 100 ug to 1 mg per ml.

All transformation experiments used ced-1(e1735); unc-31(e928)ced-3(n717) strain. unc-31 was used as a marker for co-transformation(Kim and Horvitz, 1990 supra). ced-1 was present to facilitate scoringof the ced-3 phenotype. The mutations in ced-1 block the engulfmentprocess of cell death, which makes the corpses of the dead cells persistmuch longer than in wild-type animals (Hedgecock et al., Science220:1277-1280 (1983)). The ced-3 phenotype was scored as the number ofdead cells present in the head of young L1 animals. The cosmid C10D8 orthe plasmid subclones of C10D8 were mixed with C14G10(unc-31(+)-containing) at a ratio of 2:1 or 3:1 to increase the chancesthat a Unc-31(+) transformant would contain the cosmid or plasmid beingtested as well. Usually, 20-30 animals were injected in one experiment.Non-Unc F1 progeny of the injected animal were isolated three to fourdays later. About ½ to ⅓ of the non-Unc progeny transmitted the non-Uncphenotype to F2 progeny and established a transformant line. The youngL1 progeny of such non-Unc transformant were checked for the number ofdead cells present in the head using Nomarski optics.

RESULTS

Isolation of Additional ced-3 Alleles

All of the ced-3 alleles that existed previously were isolated inscreens designed to detect viable mutants displaying the Ced phenotype(Ellis and Horvitz, Cell 44:817-829 (1986)). Such screens may havesystematically missed any class of ced-3 mutations that is inviable ashomozygotes. For this reason, a scheme was designed that could isolaterecessive lethal alleles of ced-3. Four new alleles of ced-3 (n1163,n1164, n1165, n1286) were isolated in this way. Since new alleles wereisolated at a frequency of about 1 in 2500, close to the frequencyexpected for the generation of null mutations by EMS in an average C.elegans gene (Brenner, Genetics 77:71-94 (1974); Greenwald and Horvitz,Genetics 96:147-160 (1980)), and all four alleles are homozygous viable,it was concluded that the null allele of ced-3 is viable.

Mapping RFLPs near ced-3

Tc1 is a C. elegans transposable element that is thought to be immobilein the common laboratory Bristol strain and in the Bergerac strain(Emmons et al., Cell 32:55-65 (1983)). In the Bristol strain, there are30 copies of Tc1, while in the Bergerac strain, there are more than 400copies of Tc1 (Emmons et al., 1983 supra; Finney, Ph.D. thesis,Massachusetts Institute of Technology, Cambridge, Mass., 1987). Becausethe size of the C. elegans genome is small (haploid genome size 8×10⁷bp) (Sulston and Brenner, Genetics 77:95-104 (1976)), a polymorphism dueto Tc1 between the Bristol and Bergerac strains would be expected tooccur about once every 200 kb. Restriction fragment length polymorphisms(RFLPs) can be used as genetic markers and mapped in a manner identicalto conventional mutant phenotypes. A general scheme has been designed tomap Tc1 elements that are dimorphic between the Bristol and Bergeracstrains near any gene of interest (Ruvkun et al., Genetics, 121:501-516(1989)). Once tight linkage of a particular Tc1 to a gene of interesthas been established, that Tc1 can be cloned and used to initiatechromosome walking.

A 5.1 kb Bristol-specific Tc1 EcoRI fragment was tentatively identifiedas containing the Tc1 closest to ced-3. This Tc1 fragment was clonedusing cosmids from a set of Tc1-containing C. elegans Bristol genomicDNA fragments. DNA was prepared from 46 such TC1-containing cosmids, andthis DNA was screened using Southern blots to identify the cosmids thatcontain a 5.1 kb EcoRI Tc1-containing fragment. Two such cosmids wereidentified: MMM-C1 and MMM-C9. The 5.1 kb EcoRI fragment was subclonedfrom MMM-C1 into pUC13 (Promega). Since both ends of Tc1 contain anEcoRV site (Rosenzweig et al., Nucleic Acids Res. 11:4201-4209 (1983)),EcoRV was used to remove Tc1 from the 5.1 kb EcoRI fragment, generatinga plasmid that contains only the unique flanking region of thisTc1-containing fragment. This plasmid was then used to map the specificTc1 without the interference of other Tc1 elements.

unc-30(e191) ced-3(n717) dpy-4(e1166)/+++ males were crossed withBergerac (EM1002) hermaphrodites, and Unc non-Dpy or Dpy non-Uncrecombinants were picked from among the F2 progeny. The recombinantswere allowed to self-fertilize, and strains that were homozygous foreither unc-30(e191) dpy-4(Bergerac) or unc-30(Bergerac) dpy-4(e1166)were isolated. After identifying the ced genotypes of these recombinantstrains, DNA was prepared from these strains. A Southern blot of DNAfrom these recombinants was probed with the flanking sequence of the 5.1kb EcoRI Tc1 fragment. This probe detects a 5.1 kb fragment in BristolN2 and a 3.4 kb fragment in Bergerac. Five out of five unc-30 ced-3dpy(+Berg) recombinants, and one of one unc-30(+Berg) ced-3 dpy-4recombinants showed the Bristol pattern. Nine of ten unc-30(+Berg) dpy-4recombinants showed the Bergerac pattern. Only one recombinant ofunc-30(+Berg) dpy-4 resulted from a cross-over between ced-3 and the 5.1kb Tc1 element. The genetic distance between ced-3 and dpy-4 is 2 mapunits (mu). Thus, this Tc1 element is located 0.1 mu on the right sideof ced-3.

Cosmids MMM-C1 and MMM-C9 were used to test whether any previouslymapped genomic DNA cosmids overlapped with these two cosmids. A contigof overlapping cosmids was identified that extended the cloned regionnear ced-3 in one direction.

To orient MMM-C1 with respect to this contig, both ends of MMM-C1 weresubcloned and these subclones were used to probe the nearest neighboringcosmid C48D1. The “right” end of MMM-C1 does not hybridize to C48D1,while the “left” end does. Therefore, the “right” end of MM-C1 extendsfurther away from the contig. To extend this contig, the “right” end ofMMM-C1 was used to probe the filters of two cosmid libraries (Coulson etal. , Proc. Natl. Acad. Sci. USA 83:7821-7825 (1986)). One clone, Jc8,was found to extend MMM-C1 in the opposite direction of the contig.

RFLPs between the Bergerac and Bristol strains were used to orient thecontig with respect to the genetic map. Bristol (N2) and Bergerac(EM1002) DNA was digested with various restriction enzymes and probedwith different cosmids to look for RFLPs. Once such an RFLP was found,DNA from recombinants of the Bristol and Bergerac strains between ced-3and unc-26, and between unc-30 and ced-3 was used to determine theposition of the RFLP with respect to ced-3.

The “right” end of Jc8, which represents one end of the contig, detectsan RFLP (nP33) when N2 and EM1002 DNA was digested with HindIII. ASouthern blot. of DNA from recombinants between three ced-3(+Berg)unc-26 was probed with the “right” end of Jc8. Three of three +Bergunc-26 recombinants showed the Bristol pattern, while two of two ced-3unc-26(+Berg) recombinants showed the Bergerac pattern. Thus, nP33mapped very close or to the right side of unc-26.

The “left” end of Jc8 also detects a HindIII RFLP (nP34). The sameSouthern blot was reprobed with the Jc8 “left” end. Two of the two ced-3unc-26(+Berg) recombinants and two of the three ced-3(+Berg) unc-26recombinants showed the Bergerac pattern. One of the three ced-3(+Berg)unc-26 recombinants showed the Bristol pattern. The genetic distancebetween ced-3 and unc-26 is 0.2 mu. Thus, nP34 was mapped between ced-3and unc-26, about 0.1 mu on the right side of ced-3.

The flanking sequence of the 5.1 kb EcoRI Tc1 fragment (named nP35) wasused to probe the same set of recombinants. Two of three ced-3(+Berg)unc-26 recombinants and two of two ced-3 unc-26(+Berg) recombinantsshowed the Bristol pattern. Thus, nP35 was also found to be locatedbetween ced-3 and unc-26, about 0.1 mu on the right side of ced-3.

A similar analysis using cosmid T10H5 which contains the HindIII RFLP(nP36), and cosmid B0564, which contains a HindIII RFLP (nP37), showedthat nP36 and nP37 mapped very close or to the right of unc-30.

These experiments localized the ced-3 gene to an interval of threecosmids. The positions of the RFLPs, and of ced-3, unc-30 and unc-26 onchromosome IV, and their relationships to the cosmids are shown in FIG.9. It was has been further demonstrated by microinjection that cosmidsC37G8 and C33F2 carry the unc-30 gene (John Sulston, personnelcommunication). Thus, the region containing the ced-3 gene was limitedto an interval of two cosmids. These results are summarized in FIG. 9.

Complementation of ced-3 by Germline Transformation

Cosmids that were candidates for containing the ced-3 gene weremicroinjected into a ced-3 mutant to see if they rescue the mutantphenotype. The procedure for microinjection was that of A. Fire (EMBO J.5:2673-2680 (1986)) with modifications. unc-31, a mutant defective inlocomotion, was used as a marker for cotransformation (Kim and Horvitz,Genes & Dev. 4:357-371 (1990)), because the phenotype of ced-3 can beexamined only by using Nomarski optics. Cosmid C14G10 (containingunc-31(+)) and a candidate cosmid were coinjected into ced-l e1375);unc-31 (e928) ced-3 (n717) hermaphrodites, and F1 non-Unc transformantswere isolated to see if the non-Unc phenotype could be transmitted andestablished as a line of transformants. Young L1 progeny of suchtransformants were examined for the presence of cell deaths usingNomarski optics to see whether the ced-3 phenotype was suppressed.Cosmid C14G10 containing unc-31 alone does not rescue ced-3 activitywhen injected into a ced-3 mutant. Table 4 summarizes the results ofthese transformation experiments.

As shown in Table 4, of the three cosmids injected (C43C9, W07H6 andC48D1), only C48D1 rescued the ced-3 phenotype (2/2 non-Unctransformants rescued the ced-3 phenotype). One of the transformants,nEX2, appears to be rescued by an extra-chromosomal array of injectedcosmids (Way and Chalfie, Cell 54:5-16 (1988)), which is maintained asan unstable duplication, since only 50% of the progeny of a non-UncCed(+) animal are non-Unc Ced(+). Since the non-Unc Ced(+) phenotype ofthe other transformant (nIS1) is transmitted to all of its progeny, itis presumably an integrated transformant. L1 ced-1 animals contain anaverage of 23 cell corpses in the head (Table 5). L1 ced-1; ced-3animals contain an average of 0.3 cell corpses in the head. ced-1;unc-31 ced-3; nIS1 and ced-1; unc-31 ced-3; nEX2 animals contain anaverage of 16.4 and 14.5 cell corpses in the head, respectively. Fromthese results, it was concluded that C48D1 contains the ced-3 gene.

In order to locate ced-3 more precisely within the cosmid C48D1, thiscosmid was subcloned and the subclones were tested for the ability torescue ced-3 mutants (Table 5). C48D1 DNA was digested with restrictionenzymes that cut rarely within the cosmid and the remaining cosmid wasself-ligated to generate a subclone. Such subclones were then injectedinto a ced-3 mutant to look for complementation; young L1 non-Uncprogeny of the transformants were examined using Nomarski optics for thepresence of cell death in the head. When C48D1 was digested with BamHIand self-ligated, the remaining 14 kb subclone (named C48D1-28) wasfound to rescue the ced-3 phenotype when injected into a ced-3 mutant(FIG. 10 and Table 5). C48D1-28 was then partially digested with BglIIand self-ligated. Clones of various lengths were isolated and tested fortheir ability to rescue ced-3.

One clone, C48D1-43, which did not contain a 1.7 kb BglII fragment ofC48D1-28, was able to rescue ced-3 (FIG. 10 and Table 5). C48D1-43 wasfurther subcloned by digesting with BamHI and ApaI to isolate a. 10 kbBamHI-ApaI fragment. This fragment was subcloned into pBSKII+ togenerate pJ40. pJ40 can restore ced-3+ phenotype when microinjected intoa ced-3 mutant. pJ40 was subcloned by deleting a 2 kb BglII-ApaIfragment to generate pJ107. pJ107 was also able to rescue the ced-3phenotype when microinjected into a ced-3 mutant. Deletion of 0.5 kb onthe left side of pJ107 could be made by ExoIII digestion (as inpJ107del28 and pJ107del34) without affecting ced-3 activity; in fact,one transgenic line, nEX17, restores full ced-3 activity. However, theced-3 rescuing ability was significantly reduced when 1 kb was deletedon the left side of pJ107 (as in pJ107del12 and pJ107del27), and theability was completely eliminated when a 1.8 kb SalI-BglII fragment wasdeleted on the right side of pJ107 (as in pJ55 and pJ56), suggestingthat this SalI site is likely to be in the ced-3 coding region. Fromthese experiments, ced-3 was localized to a DNA fragment of 7.5 kb.These results are summarized in FIG. 10 and Table 5.

ced-3 Transcript

pJ107 was used to probe a Northern blot of N2 RNA and detected a band of2.8 kb. Although this transcript is present in 12 ced-3 mutant animals,subsequent analysis showed that all 12 ced-3 mutant alleles containmutations in the genomic DNA that codes for this mRNA (see below), thusestablishing this RNA as a ced-3 transcript.

The developmental expression pattern of ced-3 was determined byhybridizing a Northern blot of RNA from animals of different stages(eggs, L1 through L4 larvae and young adult) with the ced-3 cDNAsubclone pJ118. Such analysis revealed that the ced-3 transcript is mostabundant during embryonic development, which is the period when mostprogrammed cell deaths occur, but it was also detected during the L1through L4 larval stages and is present in relatively high levels inyoung adults. This result suggests that ced-3 is not only expressed incells undergoing programmed cell death.

Since ced-3 and ced-4 are both required for programmed cell death in C.elegans, one of the genes might-act as a regulator of transcription ofthe other gene. To examine if ced-4 regulates the transcription ofced-3, RNA was prepared from eggs of ced-4 mutants (n1162, n1416, n1894,and n1920), and a Northern blot was probed with the ced-3 cDNA subclonepJ118. The presence of RNA in each lane was confirmed with an actin Iprobe. Such an experiment showed that the level of ced-3 transcript isnormal in ced-4 mutants. This indicates that ced-4 is unlikely to be atranscriptional regulator of ced-3.

Isolation of a ced-3 cDNA

To isolate cDNA of ced-3, pJ40 was used as a probe to screen a cDNAlibrary of N2 (Kim and Horvitz, Genes & Dev. 4:357-371 (1990)). SevencDNA clones were isolated. These cDNAs can be divided into two groups:one is 3.5 kb and the other 2.5 kb. One cDNA from each group wassubcloned and analyzed further. pJ85 contains the 3.5 kb cDNA.Experiments showed that pJ85 contains a ced-3 cDNA fused to an unrelatedcDNA; on Northern blots of N2 RNA, the pJ85 insert hybridizes to two RNAtranscripts, and on Southern blots of N2 DNA, pJB5 hybridizes to morethan one band than pJ40 (ced-3 genomic DNA) does. pJ87 contains the 2.5kb cDNA. On Northern blots, pJ87 hybridizes to a 2.8 kb RNA and onSouthern blots, it hybridizes only to bands to which pJ40 hybridizes.Thus, pJ87 contains only ced-3 cDNA.

To show that pJ87 does contain the ced-3 cDNA, a frameshift mutation wasmade in the Sa1I site of pJ40 corresponding to the SalI site in the pJ87cDNA. Constructs containing the frameshift mutation failed to rescue theced-3 phenotype when microinjected into ced-3 mutant animals, suggestingthat ced-3 activity has been eliminated.

ced-3 Sequence

The DNA sequence of pJ87 was determined (see FIG. 4; Seq. ID #18). pJ87contains an insert of 2.5 kb which has an open reading frame of 503amino acids (FIG. 4; Seq. ID #19). The 5′ end of the cDNA. contains 25bp of poly-A/T sequence, which is probably an artifact of cloning and isnot present in the genomic sequence. The cDNA ends with a poly-Asequence, suggesting that it contains the complete 3′ end of thetranscript. 1 kb of pJ87 insert is untranslated 3′ region and not all ofit is essential for ced-3 expression, since genomic constructs withdeletions of 380 bp of the 3′ end can still rescue ced-3 mutants (pJ107and its derivatives, see FIG. 10).

To confirm the DNA sequence obtained from the ced-3 cDNA and to studythe structure of the ced-3 gene, the genomic sequence of the ced-3 genein the plasmid pJ107 was determined (FIG. 4; Seq. ID #18). Comparison ofthe ced-3 genomic and cDNA sequences revealed that the ced-3 gene hasseven introns that range in size from 54 bp to 1195 bp (FIG. 5A). Thefour largest introns, as well as sequences 5′ of the start codon, werefound to contain repetitive elements. Five types of repetitive elementswere found, some of which have been previously characterized innon-coding regions of other C. elegans genes such as fem-1 (Spence etal., Cell 60:981-990 (1990)), lin-12 (J. Yochem, personalcommunication), and myoD (Krause et al., Cell 63:907-919 (1990)) (FIG.4). Of these, repeat 1 was also found in fem-1 and myoD, repeat 3 inlin-12 and fem-1, repeat 4 in lin-12, and repeats 2 and 5 were novelrepetitive elements.

A combination of primer extension and PCR amplification was used todetermine the location and nature of the 5′ end of the ced-3 transcript.Two primers (Pex1 and Pex2) were used for the primer extension reaction.The Pex1 reaction yielded two major bands, whereas the Pex2 reactiongave one band. The Pex2 band corresponded in size to the smaller bandfrom the Pex1 reaction, and agreed in length with a possible transcriptthat is trans-spliced to a C. elegans splice leader (Bektesh, Genes &Dev., 2:1277-1283 (1988)) at a consensus splice acceptor at position2166 of the genomic sequence (FIG. 4). The nature of the larger Pex1band is unclear.

To confirm the existence of this trans-spliced message in wild-typeworms, total C. elegans RNA was PCR amplified using the SL1-Log5 andSL2-Log5 primer pairs, followed by a reamplification using theSL1-oligo10 and SL2-Oligo10 primer pairs. The SL1 reaction yielded afragment of the predicted length. The identity of this fragment wasconfirmed by sequencing. Thus, at least some, if not most, of the ced-3transcript is trans-spliced to SL1. Based on this result, the startcodon of the ced-3 message was assigned to the methionine encoded atposition 2232 of the genomic sequence (FIG. 4).

The DNA sequences of 12 EMS-induced ced-3 alleles were also determined(FIG. 4 and Table 3). Nine of the 12 are missense mutations. Two of the12 are nonsense mutations, which might prematurely terminate thetranslation of ced-3. These nonsense ced-3 mutants confirmed that theced-3 gene is not essential for viability. One of the 12 mutations is analteration of a conserved splicing acceptor G, and another has a changeof a 70% conserved C at the splice site, which could also generate astop codon even if the splicing is correct. Interestingly, theseEMS-induced mutations are in either the N-terminal quarter orC-terminal-half of the protein. In fact, 9 of the 12 mutations occurwithin the region of ced-3 that encodes the last 100 amino acids of theprotein. Mutations are notably absent from the middle part of the ced-3gene (FIG. 5).

Ced-3 Protein Contains A Region Rich in Serines

The Ced-3 protein is very hydrophilic and no significantly hydrophobicregion can be found that might be a trans-membrane domain (FIG. 6). TheCed-3 protein is rich in serine. From amino acid 78 to amino acid 205 ofthe Ced-3 protein, 34 out of 127 amino acids are serine. Serine is oftenthe target of serine/threonine protein kinases (Edelman, Ann. Rev.Biochem. 56:567-613 (1987)). For example, protein kinase C canphosphorylate serines when they are flanked on their amino and carboxylsides by basic residues (Edelman, 1987 supra). Four of the serines inthe Ced-3 protein are flanked by arginines (FIG. 4). The same serineresidues might also be the target of related Ser/Thr kinases.

To identify the functionally important regions of the Ced-3 protein,genomic DNAs containing the ced-3 genes from two related nematodespecies, C. briggsae and C. vulgaris were cloned and sequenced (FIG. 7;Seq. ID #20 and 21). Sequence comparison of the three ced-3 genes showedthat the non-serine-rich region of the proteins is highly conserved. InC. briggsae and C. vulgaris, many amino acids in the serine-rich regionare dissimilar compared to the C. elegans Ced-3 protein (FIG. 7). Itseems that what is important in the serine-rich region is the overallserine-rich feature rather than the exact amino acid sequence.

This hypothesis is also supported by analysis of ced-3 mutations in C.elegans: none of the 12 EMS-induced mutations is in the serine-richregion, suggesting that mutations in this region might not affect thefunction of the Ced-3 protein and thus, could not be isolated in thescreen for ced-3 mutants.

TABLE 1 Rescue of the Ced-4 Phenotype by Germline Transformation No. DNAAvg. No. Cell Animals Genotype Injected Corpses (L1 Head) Scored ced-1;ced-4; C10D8; 9.4 10 unc-31; nEx1 C14G10 ced-1; ced-4; C10D8-5 11.5 10unc-31; nEx7 C14G10 ced-1; ced-4 C10D8-5 11.5 10 unc-31; nEx8 C14G10ced-1 None 23 20 ced-1; ced-4 None 0.6 20

TABLE 2 Sites of Mutations in the ced-4 Gene Allele Mutation NucleotideCodon Consequence n1162 C to T 1131 40 Q to ochre (TAA) n2274 C to T1428 139 R to opal (TGA) n1920 & G to A 1744 first base of 5′ Alteredsplicing n2247 splice donor of intron 3 n2273 G to A 1929 first base of3′ Altered splicing splice acceptor of intron 3 n1948 T to A 2117 258 Ito N n1947 C to T 2128 262 Q to amber n1894 G to A 3131 401 W to opal(TGA) Nucleotide and codon positions correspond to the numbering in FIG.1.

TABLE 3 Sites of Mutations in the ced-3 Gene Allele Mutation NucleotideCodon Consequence n1040 C to T 2310 27 L to F n718  G to A 2487 65 G toR n2433 G to A 5757 360 G to S n1164 C to T 5940 403 Q to terminationn717  G to A 6297 — Splice acceptor loss n1949 C to T 6322 412 Q totermination n1286 G to A 6342 428 W to termination n1129 C to T 6434 449A to V n1165 C to T 6434 449 A to V n2430 C to T 6485 466 A to V n2426 Gto A 6535 483 E to K n1163 C to T 7020 486 S to F Nucleotide and codonpositions correspond to the numbering in FIG. 4.

TABLE 4 Summary of Transformation Experiments Using Cosmids in the ced-3Region Cosmid No. of non-Unc Ced-3 injected transformants phenotypeStrain name C43C9; C14G10 1 − MT4302 W07H6; C14G10 3 − MT4299 − MT4300 −MT4301 C48D1; C14G10 2 + MT4298 + MT4303 Animals injected were ofgenotype: ced-1(e1735); unc-31(e929) ced-3(n717).

TABLE 5 The expression of ced-3(+) transformants Average No. No. celldeaths Animals Genotype DNA injected in L1 head scored ced-1 — 23 20ced-1; ced-3 — 0.3 10 ced-1; nIS1 C48D1; 16.4 20 unc-31 ced-3 C14G10ced-1; unc-31 14.5 20 ced-3; nIS1/+ ced-1; unc-31 C48D1; 13.2 10/14ced-3; nEX2 C14G10 0  4/14 ced-1; unc-31 C48D1-28; 12  9/10 ced-3; nEX10C14G10 0 1 of 10 ced-1; unc-31 C48D1-28; 12 10 ced-3; nEX9 C14G10 ced-1;unc-31 C48D1-43 16.7 10/13 ced-3; nEX11 C14G10 Abnormal cell  3/13deaths ced-1; unc-31 pJ40; C14G10 13.75 4/4 ced-3; nEX13 ced-1; unc-31pJ107de128, 23 12/14 ced-3; nEX17 pJl07de134 0  2/14 C14G10 ced-1;unc-31 pJ107de128, 12.8  9/10 ced-3; nEX18 pJ107de1134 0  1/10 C14G10ced-1; unc-31 pJ107de128, 10.6 5/6 ced-3; nEX19 pJ107de134 0 1/6 G14G10ced-1; unc-31 pJ107de112, 7.8 12/12 ced-3; nEX16 pJ107de127 C14G10Alleles of the genes used are ced-1(e1735), unc-31(e928), andced-3(n717).Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such. equivalents areintended to be encompassed by the following claims. For example,functional equivalents of DNAs and RNAs may be nucleic acid sequenceswhich, through the degeneracy of the genetic code, encode the sameproteins as those specifically claimed. Functional equivalents ofproteins may be substituted or modified amino acid sequences, whereinthe substitution or modification does not change the activity orfunction of the protein. A “silent” amino acid substitution, such that achemically similar amino acid (e.g., an acidic amino acid with anotheracidic amino acid) is substituted, is an example of how a functionalequivalent of a protein can be produced. Functional equivalents ofnucleic acids or proteins can also be produced by deletion ofnonessential sequences.

1. An isolated protein comprising the amino acid sequence of SEQ IDNO.:19.
 2. The isolated polypeptide of claim 1, wherein said polypeptideconsists of the amino acid sequence of SEQ. ID NO.:19.